The present invention provides for improved riboflavin production. In particular, the invention provides transformed yeast strains and processes for producing riboflavin.
Derivatives of riboflavin (the flavocoenzymes FMN and FAD) are universally required for redox reactions in all cellular organisms. Riboflavin (vitamin B2) is produced by all plants and by many microorganisms (Demain A. L. Riboflavin over synthesis. Ann. Rev. Microbiol. 1972, 26, 369). The compound is not produced in vertebrates. Riboflavin is therefore an essential nutrient for man and animals.
Riboflavin may be produced by chemical synthesis and by various fermentation procedures using, for example, strains of Bacillus (e.g., Bacillus subtilis), the ascomycetes Ashbya gossypii and Eremothecium ashbyi (Demain A. L. Riboflavin Over Synthesis. Ann. Rev. Microbiol. 1972, 26, 369 and Mitsuda H, Nakajima K., Effects of 8-azaguanine on Riboflavin Production and on the Nucleotide Pools in Non-Growing Cells of Eremothecium ashbyii. J. Nutr Sci Vitaminol (Tokyo) 1973; 19(3):215-227), various yeast strains such as Candida guilliermondii, Candida famata (F. W. Tanner, Jr., C. Vojnovich, J. M. Van Lanen. Riboflavin Production by Candida Species. Nature, 1945, 101 (2616):180-181) and related strains, as well as other microorganisms.
The pathway of riboflavin biosynthesis in yeast is shown in FIG. 1. The recursors for the biosynthesis of riboflavin are guanosine triphosphate (GTP) and ribulose 5-phosphate. One mole of GTP and two moles of ribulose 5-phosphate are required to biosynthetically generate one mole of riboflavin.
In the yeast Saccharomyces cerevisiae, the biosynthesis of riboflavin requires at least six genes, specifically the genes RIB1, RIB2, RIB3, RIB4, RIB5 and RIB7 (Oltmanns O., Bacher A., Lingens F. and Zimmermann F. K., Biochemical and Genetic Classification of Riboflavin Deficient Mutants of Saccharomyces cerevisiae. Mol. Gen. Genet. 1969, 105, 306). In C. guilliermondii, the biosynthesis of riboflavin has also been reportedly shown to require the products of at least six genes, specifically the genes RIB1, RIB2, RIB3, RIB4, RIB5 and RIB6 (Shavlovskyy, G. M., Sibirnyy, A. A., Kshanovs""ka, B. V. Genetical classification of C. guilliermondii riboflavin auxotroph mutants. Genetika 15, 1561-1568, 1979). The enzymes specified by these C. guilliermondii genes and their roles in the biosynthetic pathway are also summarized in FIG. 1 and Table 1. In contrast to the situation in B. subtilis, the riboflavin biosynthetic genes are not clustered in the eucatyotes S. cerevisiae and C. guilliermondii.
The initial step in the riboflavin biosynthetic pathway is the opening of the imidazole ring of GTP catalyzed by the enzyme, GTP cyclohydrolase II. The product of this enzyme has been reported to be 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5xe2x80x2-phosphate. This intermediate is converted to 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione by a sequence of side chain reduction, ring deamination and dephosphorylation reactions. The enzyme involved in the dephosphorylation of 5-amino-6-ribitylamino 5xe2x80x2-phosphate is still unknown.
The conversion of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione to 6,7-(1dimethyl-8-ribityllumazine by the enzyme, 6,7-dimethyl-8-ribityllumazine synthase, requires a second substrate, 3,4-dihydroxy-2-butanone 4-phosphate, which is obtained from ribulose 5-phosphate by the catalytic action of 3,4-dihydroxy-2-butanone-4-phosphate synthase. Finally, 6,7-dimethyl-8-ribityllumazine is converted to riboflavin by a dismutation reaction catalyzed by riboflavin synthase. The sequence of the RIB1 gene directing the synthesis of GTP cyclohydrolase II, the initial enzyme of the riboflavin pathway, reportedly has been established in the yeast, C. guilliermondii (Liauta-Teglivets, O., Hasslacher, M., Boretskyy, Y., Kohlwein, S. D., Shavlovskii, G. M. Molecular cloning of the GTP cyclohydrolase. Structural gene RIB1 of Pichiia guilliermondii involved in riboflavin biosynthesis. Yeast 11, 945-952, 1995).
Recombinant strains of Bacillus subtilis used in the production of riboflavin by fermentation have been reportedly described, e.g. in EP 405 370. These strains carry the riboflavin operon under the control of a strong promoter directing the production of the cognate enzymes. The gene constructs of the riboflavin operon under the control of a strong; promoter may be present at one or several different locations on the B. subtilis chromosome. The incorporation of an additional gene of the riboflavin pathway under the control of a strong promoter at a separate locus on the B. subtilis chromosome reportedly has also been shown to increase the yield of riboflavin obtained by fermentation. See, EP 821 063.
Whereas the production of riboflavin by native strains of yeasts such as C. guilliermondii have been reported, these reports suffer from the drawback that relatively small amounts of riboflavin are produced therefrom. Heretofore, recombinant DNA technology has not been applied to over-express riboflavin biosynthetic genes in C. guillietmondii or in related flavinogenic yeasts.
The invention is a recombinant DNA construct used to transform yeast strains which over-produce riboflavin.
More specifically, the present invention provides a yeast strain transformed by a recombinant DNA sequence such as a DNA sequence which, upon expression in a suitable host cell, encodes at least one polypeptide with riboflavin biosynthetic activity and which DNA sequence is transcriptionally linked to a promoter that is functional in such a yeast strain.
Another embodiment of the invention is a transformed yeast strain which belongs to the group of flavinogenic yeasts which over-produce riboflavin under conditions of iron starvation.
A further embodiment of the present invention is a transformed yeast strain wherein the polypeptide encoding DNA sequence is isolated or derived from yeast, preferably a flavinogenic yeast which over-produces riboflavin under conditions of iron starvation, such as Candida, e.g. Candida guilliermondii or Candida famata. 
Another embodiment of the present invention is a yeast strain wherein the polypeptide encoding DNA sequence encodes a protein with GTP cyclohydrolase II activity and is selected from the following DNA sequences:
a) the DNA sequence as shown in FIG. 4 or its complementary strand;
b) DNA sequences which hybridize under standard conditions to the protein coding regions of the DNA sequences defined in (a) or fragments thereof; and
c) DNA sequences which, but for the degeneracy of the genetic code, would hybridize to the DNA sequences defined in (a) and (b).
Another embodiment is a yeast strain transformed with a DNA construct as set forth above, wherein the promoter is the TEF S. cerevisiae promoter.
Another embodiment is a process for producing riboflavin by culturing a yeast strain as set forth above under suitable culture conditions to express riboflavin and then isolating the riboflavin from the medium or the yeast strain.
Another embodiment of the present invention is a process for producing riboflavin by mixing the isolated riboflavin with one or more suitable food or feed ingredients to form a food or feed composition.
Another embodiment is an isolated and purified DNA molecule which encodes a polypeptide with riboflavin biosynthetic activity isolated from a yeast strain and which encodes a polypeptide with riboflavin biosynthetic activity that is encoded by a nucleotide sequence including the DNA sequence in FIG. 4 and functionally equivalent fragments, mutants and derivatives thereof.
Another embodiment is a process for producing a yeast cell which over-expresses riboflavin. This process includes (a) identifying a nucleotide sequence coding for a polypeptide having riboflavin biosynthetic activity; (b) incorporating the nucleotide sequence into an expression cassette; (c) transforming a yeast cell culture with the expression cassette; (d) selecting a transformant which over-expresses riboflavin; (e) culturing the transformant in a culture medium; and (f) recovering riboflavin from the transformant and/or the culture medium.